Nuclei were sorted at the Flow Cytometry core at the University of Chicago on a BD FACSAria or BD FACSAria Fusio equipped with a 96-well-plate holder. To obtain individual and intact nuclei gates were set on forward and side scatter to exclude aggregates and debris. DAPI/PacBlue channel or Violet 450/500 channel were usedto excited the Hoechst 33342 DNA dye and to gate on cells with DNA content corresponding to cells in G1 phase of the cell cycle in order to maintain similar DNA content per cell and to remove potential heterogeneity attributable to cell cycle. Cells were sorted into individual wells pre-filled with 19 ul of 1x M-Digestion buffer (EZ DNA Methylation Direct Kit, Zymo Research) containing 1 mg/ml Proteinase K. Following collection, the plates were briefly spun to collect droplets that might formed during handling. Nuclei were lysed by incubating the samples at 50 C for 20 min in a PCR cycler. DNA was subjected to bisulfite conversion by adding 130 ul of freshly prepared CT Conversion reagent (EZ DNA Methylation Direct Kit, Zymo) to the lysed nuclei. Conversion was performed by denaturing the DNA at 98 C for 8 min followed by 3.5 hr incubation at 65 C. DNA isolation was performed using the EZ DNA Methylation Direct Kit (Zymo Research) following the manufacturer’s instruction with the modification that the DNA was eluted in only 8 ul of elution buffer.
Ez dna methylation direct kit
Manufactured by Zymo Research
Sourced in United States, Germany
The EZ DNA Methylation-Direct Kit is a product offered by Zymo Research. It is designed for the conversion of DNA samples for bisulfite sequencing, which is a method used to analyze DNA methylation patterns. The kit provides a rapid and efficient way to convert unmethylated cytosine residues to uracil, while leaving methylated cytosines unchanged, allowing for the detection of DNA methylation status.
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Topo ta cloning kit, by Thermo Fisher Scientific (11 mentions)
Pgem t vector, by Promega (10 mentions)
Dneasy blood tissue kit, by Qiagen (9 mentions)
Pyromark pcr kit, by Qiagen (8 mentions)
Pmd18 t vector, by Takara Bio (7 mentions)
Qiaquick gel extraction kit, by Qiagen (7 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (7 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (7 mentions)
Pyromark q24 system, by Qiagen (6 mentions)
Dneasy blood and tissue kit, by Qiagen (6 mentions)
Zymotaq premix, by Zymo Research (6 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (5 mentions)
Biotaq hs dna polymerase, by Meridian Bioscience (5 mentions)
Imprint dna modification kit, by Merck Group (5 mentions)
Pmd19 t vector, by Takara Bio (5 mentions)
Hiseq 2500, by Illumina (4 mentions)
Pico methyl seq library prep kit, by Zymo Research (4 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Spriselect beads, by Beckman Coulter (4 mentions)
298 protocols using ez dna methylation direct kit
1
Single-Cell DNA Methylation Profiling
2
Bisulfite Conversion of Genomic DNA
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For this step, the EZ DNA Methylation-Direct kit (Zymo Research, Irvine, CA, USA) was used, and the protocol provided by the company was followed. The EZ DNA Methylation-Direct™ kit requires a DNA extract input of 20 μL, with a concentration of 250–500 ng/20 μL. The samples’ extracts and controls were diluted or concentrated to obtain a concentration as close as possible to 500 ng/20 μL. Alongside the samples, two control DNAs, one methylated and one non-methylated (HCT116 DKO methylated and non-methylated DNA by Zymo Research) were bisulfite-converted, so that they could be analyzed following the same protocol, in order to check for a correct bisulfite conversion.
3
Bisulfite Conversion of Genomic DNA
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A CpG modification Kit (EZ DNA methylation-DirectTM Kit, Zymo Research, CA) was used to convert genomic DNA according to the manufacturer’s protocol and as we previously described1 .
4
Bisulfite Sequencing of NTSR1 in CRC Cell Lines
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CRC cell lines (HCA7, HCT116, LIM1215, RKO, SW480, and T84) and normal samples (DNA from normal colonic mucosa (Dr. P. Set, Amsbio, Abingdon, United Kingdom) and blood-derived healthy control DNA (Promega, Madison, WI, USA) were bisulfite converted using the EZ DNA Methylation-DirectTM Kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s protocol. Methylation-unbiased primers (Supplementary Figure S1 and Supplementary Table S6 ) were used to amplify the bisulfite converted DNA samples, and amplified products were sequenced directly or after cloning. NTSR1 was selected for the cloning experiment (Supplementary Figure S2 ). Amplified DNA samples were cloned into a pCRC2.1 TOPO vector using the TOPO TA Cloning System (Invitrogen, Carlsbad, CA, USA) and One Shot Electrocompetent E. coli (Invitrogen, Carlsbad, CA, USA). Resulting white colonies were used for the purification of DNA and were sequenced, totaling 14–30 sequences per amplification product. The frequency of methylated sites was calculated and compared to MS-MLPA results (Supplementary Figure S2 ).
5
Bisulfite Sequencing of ASCT2 Promoter
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To convert non-methylated cytosine to uracil leaving 5-methylcytosines unmodified, 500 ng of DNA samples were treated using the EZ DNA Methylation-DirectTM Kit (Zymo Research, Irvine, CA) according to the manufacturer's protocol; and utilized as a template for nested PCR to amplify the region within the ASCT2 promoter. PCR products were gel-purified and inserted into the pGEM-T vector (Promega, Beijing, China). The transformed clones (at least 20 from each sample) were used to purify the plasmids, which were sequenced in the Beijing Genomics Institute (BGI, Beijing, China).
6
Genome-wide DNA Methylation Analysis by Reduced Representation Bisulfite Sequencing
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Chloroform was added (1:2 ratio) to the Trizol lysates before centrifuging at 4 °C for 15 min at 13000 x g. DNA was isolated from the phase separated organic layer. DNA was then purified using the Clean and Concentrator kit (Zymo Research, Irvine, CA, USA) and resuspended in a volume of 20 μl. EpiQuest libraries, which were designed to be non-directional, were prepared from 500 ng ultrapure genomic DNA. The DNA was digested with 60 units of TaqI and 30 units of MspI (NEB) sequentially. Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled-in and 3′-terminal-A extended. Ligation to pre-annealed barcoded adaptors containing 5′-methyl-cytosine was performed using the Illumina DNA preparation kit and protocol. Purified, adaptor-ligated fragments were bisulphite treated using the EZ DNA Methylation-Direct(tm) Kit (Zymo Research). Preparative-scale PCR was performed and DNA Clean and Concentrator-purified PCR products were subjected to a final size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices containing adaptor-ligated fragments of 130–210 or 210–460 bp in size were excised. Barcoded libraries were sequenced across 2 lanes of an Illumina HiSeq 2000, generating 50 bp paired end reads.
7
Bisulfite Conversion and COBRA Analysis
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Bisulfite conversion and COBRA were performed according to previous study (Chen et al. 2014 (link)). Briefly, for bisulfite modification, genomic DNA from different tissues and germ cells was treated with the CpGenome™ Turbo Bisulfite Modification Kit (Millipore) and EZ DNA Methylation-Direct TM Kit (Zymo Research), respectively. Primer sequences used for BSP are described in Additional file 1 : Table S1. The PCR products were subjected to T vector cloning (positive clones, n = 10) and sequencing analysis, which showed heterozygosity at the (C/T) peak of Nnat. BSP products were also digested by a restriction enzyme BstUI (Thermo Scientific, MA, USA) for COBRA analysis. DNAMAN (LynnonBiosoft) and the online software tools MethOrimer and BiQ Analyzer were used for methylation analysis in this study.
8
Methylome Profiling by PBAT-Seq
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MethylC-seq libraries were generated using post-bisulfite adapter tagging (PBAT) to avoid the bisulfite-induced loss of intact sequencing templates as described [48 (link)] with the following modifications. Briefly, genomic DNA was subjected to bisulfite treatment for 200 min with EZ DNA Methylation-DirectTM Kit (Zymo D5020). Bisulfite-treated DNA was then preamplified for two cycles with primers (5′-CCCTACACGACGCTCTTCCGATCTNNNNNN-3′) containing random hexamers and purified using the Zymo DNA Clean and Concentrator kit. Adaptor primers (5′-CAGACGTGTGCTCTTCCGATCTNNNNNN-3′) were added to preamplified products and then amplified for 12 PCR cycles with indexing primers for Illumina sequencing. Methylome libraries were purified using Beckman Coulter AMPureXP DNA beads. Libraries quality checked for fragment length between 200 and 600 bp were used for sequenced in single-read mode on an Illumina HiSeq2500 or Nextseq instrument.
9
Bisulfite Sequencing of CDKN2A CpG Island
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gDNA was extracted from 293T or HCT116 cells by a standard phenol/chloroform protocol. gDNA was subjected to bisulfite treatment using the EZ DNA Methylation-DirectTM Kit (Zymo Research, Irvine, CA, USA). Bisulfite-treated HCT116 gDNA was subjected to PCR with TaKaRa EpiTaqTM HS (for bisulfite-treated DNA) (Takara Bio, Shiga, Japan), with primers hCDKN2A-Bisul-CpG-free-F and hCDKN2A-Bisul-CpG-free-R, as shown in Table S1 , to amplify a CpG island of the human CDKN2A gene (encoding p16), which is completely CpG-methylated in one but not both alleles in HCT116 cells [24 (link)]. PCR cycles were as follows: 40 cycles of 98 °C for 10 s; 55 °C for 30 s; 72 °C for 1 min. PCR products were cloned into T-vector pMD20 (Takara Bio, Shiga, Japan) and subjected to DNA sequencing analysis. Plasmids containing CpG-methylated or unmethylated DNA-derived sequences were named pMD20_p16_M or pMD20_p16_U, respectively.
10
Bisulfite Conversion and Multiplex PCR Amplification for mtDNA Analysis
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The mtDNA was modified (non-methylated C residues converted to U) via a bisulphite conversion step, EZ DNA Methylation-DirectTM Kit (Cat. 5020 and 5021, Zymo Research).[25 ] Multiplex PCR amplification of the RsOI (Fluidigm Access ArrayTM System, BioMark, USA) generated pools of amplicons that also employed a universal forward tag common sequence 1 (CS1), and universal reverse tag common sequence 2 (CS2) (CS1= 5′-ACACTGACGACATGGTTCTACA-3′, CS2 = 5′-TACGGTAGCAGAGACTTGGTCT-3′). Each individual amplicon pool was subsequently barcoded. Two age-matched libraries, each consisting of 41 individuals, were created and purified (ZR-96 DNA Clean & Concentrator™ - ZR, Cat.# D4023) and then prepared for massively parallel sequence by Illumina next generation sequencing (NGS) using a MiSeq V2 300bp Reagent Kit (cat. # MS-102-2001), (paired-end sequencing protocol) according to the manufacturer's guidelines.
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